An early event in the response of most inflammatory cells to immunologic activation and other stimuli is the release of newly formed products (mediators) which alter the function and biochemistry of surrounding cells and tissues. The ensuing biological responses, as well as much of the pathogenesis which is attributed to inflammation and allergy, are thought to be dependent on the effects that these newly-formed mediators have on adjacent cells within the inflammatory region.
In the last 20 years, it has become apparent that lipid mediators are among the most potent and important products which are generated during inflammatory reactions. The synthesis of most lipid mediators is initiated by the specific cleavage of complex phospholipid molecules which contain arachidonate at their sn-2 position. Arachidonic acid is predominantly found in the sn-2 position of phospholipids after redistribution by transacylases and its release by sn-2 acylhydrolases from phospholipids represents the rate-limiting step in the formation of eicosanoids (leukotrienes, prostaglandins and thromboxanes) and other hydroxylareal fatty acids. As arachidonic acid is released, it is then convened to oxygenareal derivatives by at least two enzymatic systems (lipoxygenase and/or cyclooxygenase). Concomitant with arachidonate release, lysophospholipids are formed. One of these lyso phospholipids, 1-alkyl-2-lyso-sn-glycero-3-phosphocholine, is then acetylated to form platelet-activating factor (PAF). Each of the cell types involved in the inflammatory response produce and secrete a unique subset of lipid mediators. The quantities and nature of the metabolites depend on which enzymes and precursor phospholipid pools are available to inflammatory cells.
Once lipid mediators such as PAF and eicosanoids are formed by the aforementioned pathways, they induce signs and symptoms observed in the pathogenesis of various inflammatory disorders. Indeed, the pathophysiological activity of arachidonic acid (and its metabolites) is well known to those skilled in the art. For example, these mediators have been implicated as having an important role in allergy, asthma, anaphylaxis, adult respiratory distress syndrome, reperfusion injury, inflammatory bowel disease, rheumatoid arthritis, endotoxic shock, and cardiovascular disease. Aalmon et al., Br. Meal. Bull (1978) 43:285-296; Piper et al., Ann. NY Acad. Sci. (1991) 629:112-119; Holtzman, Am. Rev. Respir. Dis. (1991) 143:188-203; Snyder, Am. J. Physiol. Cell Physiol. (1990) 259:C697-C708; Prescott et at., J. Biol. Chem. (1990) 265:17381-17384.
Similar to arachidonate products, PAF is a potent proinflammatory mediator produced by a variety of cells. In vitro, PAF stimulates the movement and aggregation of neutrophils and the release therefrom of tissue-damaging enzymes and oxygen radicals. PAF has also been implicated in activation of leukocytes, monocytes, and macrophages. These activities contribute to the actions of PAF as having (pathological) physiological activity in inflammatory and allergic responses. PAF has also been implicated in smooth muscle contraction, pain, edema, hypotensive action, increases in vascular permeability, cardiovascular disorders, asthma, lung edema, endotoxin shock, and adult respiratory distress syndrome. PAF elicits these responses either directly through its own cellular receptor(s) or indirectly by inducing the synthesis of other mediators.
Accordingly, a method which antagonises the production of free arachidonic acid, its metabolites or PAF will have clinical utility in the treatment of a variety of allergic, inflammatory and hypersecretory conditions such as asthma, arthritis, rhinitis, bronchitis and urticaria, as well as reperfusion injury arid other disease involving lipid mediators of inflammation. Many published patent applications or issued U.S. patents exist which describe various compounds having utility as PAF or eicosanoid antagonists. Such patents include U.S. Pat. Nos. 4,788,205, 4,801,598, 4,981,860, 4,992,455, 4,983,592, 5,011,847, 5,019,581 and 5,002,941.
Phospholipase A.sub.2 's (PLA.sub.2 (EC 3.1.1.4)) are responsible for the liberation of arachidonic acid from the sn-2 position of phospholipid. They are thought to play an important role in the pathogenesis of inflammation and possibly in immunological dysfunction, both as a cell associated enzyme as well as an extracellular soluble enzyme. Low molecular weight, mammalian Type II 14 kDa PLA.sub.2 has been well characterized and is known to exist in both an extracellular form in inflammatory fluids (Kramer et at., J. Biol. Chem., 264:5768-5775 (1989) and in a cell associated form (Kanda et al., Biochemical and Biophysical Research Communications, 163:42-48 (1989) and has been found in a variety of cells and tissues or extracellularly when released in response to antigenic activators or pro-inflammatory mediators such as Interleukin (IL)-1, IL-6 or tumor necrosis factor (TNF). Its presence in such inflammatory fluids, tissue exudates or serum has therefore implicated Type II-14 kDa-PLA.sub.2 's role in inflammation (Vadas, et at., (1985) Life Sci. 36, 579-587; and Seilhamer, et at., (1989) J. Biol. Chem. 264, 5335-5338). Recently, the elevated serum levels of PLA.sub.2 activity during an inflammatory insult have been attributed to cytokine induction of acute phase protein release from liver, of which the 14 kDa-PLA.sub.2 is suggested to be a part (Crowl, et al., (1991) J. Biol. Chem. 266, 2647-2651 ). In addition, soluble PLA.sub.2 activity is markedly elevated in the serum and synovial fluid of patients with rheumatoid arthritis (Stefahski et al., J. Biochem. 100:1297-303 (1986). Furthermore, increasing serum PLA.sub.2 levels have been shown to positively correlate with clinical severity (Bomalaski and Clark, Arthritis and Rheumat. 36:190-198 (1993)).
Various inhibitors of PLA.sub.2 have been described in publications and in U.S. Patents. See for instance U.S. Pat. Nos. 4,959,357; 4,933,365; 5,208,223; 5,208,244; Marshall et at., J. Rheumatology 18:1 (1991); Marshall et at., Phospholipase A.sub.2, Ed. Pyu Wong, Plenum Press, N.Y. (1990) pages 169-181; Wilkerson, et at., Eur. J. Med. Chem., 26:667, 1991; Wilkerson, Antiinflammatory Phospholipase A.sub.2 Inhibitors, Drugs of the Future, Vol. 15, No. 2 p 139-148(1990) and Bennion et al., WO 91/08737 published 27 Jun. 1991.
Bennion et at., supra, discloses compounds having the general formula R.sub.1 -CON(R.sub.5)--C(R.sub.2)H--(CH.sub.2)n--[Y(CH.sub.2)p]q--COR.sub.3 wherei n interalia, R.sub.1 is an alkyl group optionally substituted by an aryl, or hydroxy or cycloalkyl; R.sub.5 is H, aryl, alkyl or alkenyl; Y is CHR.sub.6, --CH.dbd.CH, O or S; and R.sub.3 is OH, alkoxy, or NHR.sub.31 and R.sub.31 is hydrogen, alkyl, OH, or alkoxy optionally substituted by an aryl group.
Accordingly, as PLA.sub.2 is important in the liberation of arachidoninc acid from phospholipid and may also play a role in the generation of PAF via lysophospholipid formation, inhibition of such an enzyme would be useful for the treatment of disease states caused thereby.
There are many novel forms of phospholipase A2's which have recently been discovered. For the purposes herein, members of the sn-2 acylhydrolase family of PLA2's are divided into low and high molecular weight enzymes be it from mammalian, or non-mammalian sources. Low molecular weight PLA.sub.2 's will generally have a molecular weight in the range of 12,000 to 15,000. High molecular weight will be in the range of 30,000 or 56,000 kDa to 110,000 by SDS electrophoresis analysis.
A high molecular weight, cytosolic 85 kDa PLA.sub.2 has been isolated and cloned from the human moncytic cell line, U937 (Clark et al., Proc. Natl. Acad. Sci., 87:7708-7712, 1990). The cell-associated Type II-14 kDa-PLA.sub.2 in cell lipid metabolism was thought to be the key rate limiting enzyme in lipid mediator formation, until the recent identification of this cell-associated but structurally distinct 85 kDa sn-2 acylhydrolase, (Clark, et al., supra); and Kramer, et at., (1991) J. Biol. Chem. 266, 5268-5272. Like the Type II-14 kDa enzyme, this enzyme is active at neutral pH and Ca.sup.2+ -dependent, but in contrast exhibits a preference for AA in the sn-2 position of phospholipid substrate and migrates from the cytosol to the membrane in a Ca.sup.2 +-dependent manner and is regulated by phosphorylation (Kramer et al., J. Biol. Chem., 266:5268-5272 (1991). The 85 kDa-PLA.sub.2 is also distinct from 14 kDa-PLA.sub.2 s and Ca.sup.2+ -independent PLA.sub.2 as demonstrated by different biochemical characteristics such as stability of the 85 kDa-PLA.sub.2 to DTT, instability to heat and the lack of inhibition by a phosphonate phospholipid TSA inhibitor of 14 kDa-PLA.sub.2. In addition, 85 kDa-PLA.sub.2 has been shown to possess a lysophospholipase A.sub.1 activity which is not observed with the 14 kDa-PLA.sub.2 s. The 85 kDa enzyme is similar to the myocardial Ca.sup.2+ -independent PLA2 (Bomalaski and Clark, Arthritis and Rheumat. 36:190-198. (1993)) in that Ca.sup.2+ is not required for catalysis and DTNB inhibition is observed. However, 85 kDa-PLA.sub.2 is not inhibited by the suicide inactivator bromoenol lactone, suggesting that the enzyme is distinct from the myocardial enzyme as well.
These characteristics make the 85 kDa-PLA.sub.2 a candidate for participation in the liberation of AA from phospholipid stores for subsequent metabolism to lipid mediators. Both the cytosolic 85 kDa PLA.sub.2 and a cell associated Type II 14 kDa PLA.sub.2 have been found in the human monocyte, neutrophil and platelet (Marshall and Roshak, Biochem. Cell Biol. 71:331-339 (1993)). As noted above, most of the cellular lipid mediators found elevated in a variety of inflammatory fluids are formed in response to non-pancreatic 14 kDa PLA.sub.2 action. Since arachidonate-containing phospholipids are the key precursors for a broad range of lipid mediators it would not be surprising that inflammatory cells would treat these phospholipids differently than other fatty acid-containing phospholipids. In particular, there are enzymes which control the amount of arachidonate in different phospholipid pools and these enzymes are tightly regulated to maintain arachidonate homeostasis. The movement of arachidonate into and from all phospholipids was originally thought to be exclusively by Coenzyme A-dependent acyl transferase activitites. Holub et at., Adv. Lipid Res., 16:1-125 (1978); Lands et at., In The Enzymes of Biological Membranes, ed. Martonosi, A., pp. 3-85, Plenum Press, N.Y., 1976. However, it has now been demonstrated that an enzyme, Coenzyme A-independent transcylase (CoA-IT), is involved in the movement of 20 carbon higher unsaturated fatty acids, particularly arachidonate, into particular (1-alkyl- and 1-alkenyl) phospholipid pools. These are the phospholipid pools of arachidonate that are preferentially mobilized during cell activation and utilized for eicosanoid and PAF biosynsthesis, respectively.
CoA-IT has a specificity for certain phospholipids as donor and acceptor molecules. The fatty acid transferred is long chained and unsaturated, and almost exclusively arachidonate. Other fatty acids such as the 16:0, 18:1 or 18:2 are not moved into the sn-2 position of alkyl and 1-alkenyl phospholipid pools by CoA-IT. The specificity of CoA-IT is in direct contrast to many other CoA-dependent acylation activities which acylate a wide variety of lysophospholipids with no selectivity for arachidonate.
Accordingly, as CoA-IT is involved in arachidonic acid and phospholipid metabolism, inhibition of such an enzyme would be useful for the treatment of inflammatory, allergic and hypersecretory conditions or disease states caused thereby. Therefore, a method by which CoA-IT is inhibited will consequently and preferentially decrease the arachidonate content of 1-alkyl- and 1-alkenyl-linked phospholipids and will therefore decrease the production of pro-inflammatory mediators such as free arachidonic acid, prostaglandins, leukotriene and PAF during an inflammatory response.